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Type IV secretion systems are multi-protein complexes that transfer macromolecules across the cell envelope of bacteria. Identifying the sites of interaction between the twelve proteins (VirB1–VirB11 and VirD4) that form these complexes is key to understanding their assembly and function. We have here used phage display, bacterial two-hybrid and fluorescence-based interaction assays to identify an N-terminal domain of the inner membrane protein VirB6 as a site of interaction with the envelope-spanning VirB10 protein. Our results are consistent with the notion that VirB6 acts in concert with VirB10 as well as with VirB8 during secretion system assembly and function.

Increasing evidence indicates that beta-amyloid (Abeta) oligomers rather than monomers or fibrils are the major toxic agents that specifically inhibit synaptic plasticity and long-term potentiation (LTP) in Alzheimer's disease (AD). Neutralization of Abeta oligomeric toxicity was found to reverse memory deficits. Here, we report four single-chain variable fragment (scFv) antibodies isolated from the naive human scFv library by phage display that specifically recognized Abeta oligomers but not monomers and fibrils. These conformation-dependent scFv antibodies inhibit both Abeta fibrillation and cytotoxicity and bind to the same type of eptitope displayed on the Abeta oligomers. Such scFv antibodies specifically targeting toxic Abeta oligomers may have potential therapeutic and diagnostic applications for AD.

Co-crystallization of membrane proteins with antibody fragments may emerge as a general tool to facilitate crystal growth and improve crystal quality. The bound antibody fragment enlarges the hydrophilic part of the mostly hydrophobic membrane protein, thereby increasing the interaction area for possible protein–protein contacts in the crystal. Additionally, it may restrain flexible parts or lock the membrane protein in a defined conformational state. For successful co-crystallization trials, the antibody fragments must be stable in detergents during the extended period of crystal growth and must be easily produced in amounts necessary for crystallography. Therefore, we constructed a library of antibody Fab fragments from a framework subset of the HuCAL GOLD® library (Morphosys, Munich, Germany). By combining the most stable and well expressed frameworks, VH3 and Vκ3, with the further stabilizing constant domains, a Fab library with the desired properties was obtained in a standard phage display format. As a proof of principle, we selected binders with phage display against the detergent-solubilized citrate transporter CitS of Klebsiella pneumoniae. We describe efficient methods for the immobilization of the membrane protein during selection, for ELISA screening, and for BIAcore evaluation. We demonstrate that the selected Fab fragments form stable complexes with native CitS and recognize conformational epitopes with affinities in the low nanomolar range.

We have identified Sti, the gene of a ribonuclease inhibitor from Saccharopolyspora erythraea, by using a T7 phage display system. A specific phage has been isolated from a genome library by a biopanning procedure, using RNase Sa3, a ribonuclease from Streptomyces aureofaciens, as bait. Sti, a protein of 121 amino acid residues, with molecular mass 13 059 Da, is a homolog of barstar and other microbial ribonuclease inhibitors. To overexpress its gene in Escherichia coli, we optimized the secondary structure of its mRNA by introducing a series of silent mutations. Soluble protein was isolated and purified to homogeneity. Inhibition constants of complex of Sti and RNase Sa3 or barnase were determined at pH 7 as 5×10−12 or 7×10−7, respectively.

The aim of this study was to investigate the molecular basis of human IgE–allergen interaction by screening a phage-displayed peptide library with an allergen-specific human IgE-mimicking monoclonal antibody (mAb). A mAb that reacted with major grass pollen allergens was successfully identified and shown to inhibit human IgE–allergen interaction. Biopanning of a phage-displayed random peptide library with this mAb yielded a 12 amino acid long mimotope. A synthetic peptide based on this 12-mer mimotope inhibited mAb and human IgE binding to grass pollen extracts. Our results indicate that such synthetic peptide mimotopes of allergens have potential as novel therapeutic agents.

Endothelial dysfunction plays a major role in the pathogenesis of atherosclerosis. Pro-inflammatory cytokines such as interleukin-1β and tumour necrosis factor α activate endothelial cells changing their resting phenotype to become pro-adhesive, pro-thrombotic and pro-atherogenic. Phage display in vivo biopanning has been used to identify peptide sequences that home to diseased regions of the vessel wall in low density lipoprotein receptor (LDLr) knockout mice. In LDLr knockout mice, peptide sequence determinants exhibiting organ specificity have been isolated. These sequences have applications for gene delivery, drug delivery and for improving contrast agents for vascular imaging.

Once the sequence of a genome is in hand, understanding the function of its encoded proteins becomes a task of paramount importance. Much like the biochemists who first outlined different biochemical pathways, many genomic scientists are engaged in determining which proteins interact with which proteins, thereby establishing a protein interaction network. While these interactions have evolved in regard to their specificity, affinity and cellular function over billions of years, it is possible in the laboratory to isolate peptides from combinatorial libraries that bind to the same proteins with similar specificity, affinity and primary structures, which resemble those of the natural interacting proteins. We have termed this phenomenon ‘convergent evolution’. In this review, we highlight various examples of convergent evolution that have been uncovered in experiments dissecting protein–protein interactions with combinatorial peptides. Thus, a fruitful approach for mapping protein–protein interactions is to isolate peptide ligands to a target protein and identify candidate interacting proteins in a sequenced genome by computer analysis.

The carboxy-terminal region of utrophin, like the homologous proteins dystrophin, Drp2 and dystrobrevins, contains structural domains frequently involved in protein-protein interaction. These domains (WW, EF hands, ZZ and H1-H2) mediate recognition and binding to a multicomponent complex of proteins, also known as dystrophin-associated proteins (DAPs) for their association with dystrophin, the product of the gene, mutated in Duchenne muscular dystrophy. We have exploited phage display and in vitro binding assays to study the recognition specificity of the different domains of the utrophin carboxy-terminus. We found that none of the carboxy-terminal domains of utrophin, when isolated from its structural context, selects specific ligand peptides from a phage-displayed peptide library. By contrast, panning with an extended region containing the WW, EF hands, and ZZ domain defines the consensus binding motif, PPxY which is also found in beta-dystroglycan, a component of the DAP complex that interacts with utrophin in several tissues. WW-mediated binding to PPxY peptides and to beta-dystroglycan requires the presence of the EF hands and ZZ domain. When the ZZ domain is either deleted or engaged in binding to calmodulin, the utrophin beta-dystroglycan complex cannot be formed. These findings suggest a potential regulatory mechanism by means of which the attachment of utrophin to the DAP complex can be modulated by the Ca(2+)-dependent binding of calmodulin. The remaining two motifs found in the carboxy-terminus (H1-H2) mediate the formation of utrophin-dystrobrevin hybrids but do not select ligands in a repertoire of random nonapeptides.

The Ras proteins cycle in the cell between an inactive state and an active state. In the active state, Ras signals via the switch I region to effectors like c-Raf kinase, leading to cell growth. Since Ras mutations in cancer are often associated with the presence of permanently active Ras, molecules that prevent downstream signaling may be of interest. Here, we show that by selection on the active conformation of Ras, using a recently described large phage antibody repertoire [de Haard et al. (1999) J. Biol. Chem. 274, 18218–18230], a Fab antibody (Fab H2) was identified that exclusively binds to active Ras, and not to inactive Ras. Using surface plasmon resonance (SPR) analysis, the interaction was demonstrated to be of high affinity (7.2 nM). In addition, the interaction with Ras is specific, since binding to the homologous Rap1A protein in BIAcore analysis is at least three orders of magnitude lower, and undetectable in an enzyme-linked immunosorbent assay. The antibody fragment prevents the binding of active Ras to the immobilized Ras-binding domain of c-Raf kinase (Raf-RBD) at an IC50 value of 135 nM. This value compares well to the KD of active Ras-binding to immobilized Raf-RBD using SPR, suggesting identical binding sites. Like the IgG Y13-259, which does not demonstrate preferential binding to either inactive or active Ras, Fab H2 inhibits intrinsic GTPase activity of Ras in vitro. Mapping studies using SPR analysis demonstrate that the binding sites for the antibodies are non-identical. This antibody could be used for dissecting functional differences between Ras effectors. Due to its specificity for active Ras, Fab H2 may well be more selective than previously used anti-Ras antibodies, and thus could be used for gene therapy of cancer with intracellular antibodies.

Monoclonal antibodies were raised and selected against recombinant Plantago major PmSUC2 sucrose carrier protein. Epitopes of two monoclonal antibodies (PS2-1A2 and PS2-4D4) were mapped using N-terminally truncated PmSUC2 proteins and a lambda library displaying random PmSUC2 peptides. PS2-1A2 recognizes an octapeptide close to the N-terminus of PmSUC2, PS2-4D4 binds to a decapeptide at the very C-terminus. Analyses of antibody binding to yeast protoplasts with functionally active, tagged PmSUC2 protein revealed that both epitopes are located in cytoplasmic domains of PmSUC2. These results support a model for plant sucrose transporters containing 12 transmembrane helices with the N-terminus and the C-terminus on the cytoplasmic side of the plasma membrane.

Large random hexa- and decapenta-peptide libraries were constructed and displayed on the surface of the filamentous phagemid pComb8. Panning of the hexa-peptide library on immobilized plasminogen activator inhibitor 1 (PAI-1) specifically selected a minor fraction of concatemers, indicating that binding to PAI-1 requires an extended amino acid sequence. Accordingly, the decapenta-peptide library exclusively yielded PAI-1 binding peptides of 15 amino acid residues. None of these phage-bound peptides prevented the interaction between PAI-1 and its target serine protease urokinase (u-PA). To isolate peptides that block the interaction between PAI-1 and u-PA, phages bound to immobilized PAI-1 were eluted by incubation with u-PA. Remarkably, this procedure resulted in elution of a unique phage type that harbors a concatemer of decapentamers, consisting of 49 amino acid residues with no obvious similarity to the primary sequence of PAI-1 or u-PA.

The human proteinase inhibitor, alpha2-macroglobulin (a2-M), inhibits a large number of proteinases. Alpha2-M-proteinase complexes are rapidly cleared from the circulation by binding to a cellular receptor (alpha2-M-R/LRP) via the receptor binding domain (RBD) which is made up of a 20 kDa C-terminal stretch of the 180 kDa monomer of the inhibitor. A monoclonal antibody (mab alpha-1) has been described which reacts with the receptor-recognizable form of the inhibitor, the so called transformed alpha2-M (a2-Mt). By screening of a phage display library an epitope in the RBD of the inhibitor was identified that reacts with mab alpha-1. Out of 25 phage clones a heptapeptide sequence (S-x1-x2-D-x3-x4-K) was obtained containing identical amino acids in three positions. A consensus peptide (S-R-S-D-P-P-K) was synthesized and found to displace alpha2-Mt from binding to mab alpha-1 and to receptor. The specificity of competition was demonstrated by a reversed peptide and a control antibody. By structural comparison it was found that the consensus heptapeptide mimics a discontinuous conformationally constrained epitope present in the RBD of the inhibitor. This is the first report describing the detection of discontinuous epitopes by phage display using a short linear peptide.

Functional heavy-chain γ-immunoglobulins lacking light chains occur naturally in Camelidae. We now show the feasibility of immunising a dromedary, cloning the repertoire of the variable domains of its heavy-chain antibodies and panning, leading to the successful identification of minimum sized antigen binders. The recombinant binders are expressed well in E. coli, extremely stable, highly soluble, and react specifically and with high affinity to the antigens. This approach can be viewed as a general route to obtain small binders with favourable characteristics and valuable perspectives as modular building blocks to manufacture multispecific or multifunctional chimaeric proteins.

Libraries of monovalent display-phage expressing mutant human B-type natriuretic peptide (hBNP) were used to identify variants that preferentially bind natriuretic peptide receptor-A (NPR-A) compared to receptor-C (NPR-C). Position 19 was a significant determinant of receptor specificity for hBNP display phage. The synthetic hBNP variant S19R had a 265-fold improved NPR-A binding over NPR-C, analogous to the atrial natriuretic peptide (ANP) specificity mutation G16R. Mutation of the last three residues of the hBNP disulfide ring, G23F/L24W/G25R, resulted in about 9-fold improved selectivity. The analogous mutations in ANP decreased NPR-A binding, suggesting divergence in the mechanism of NPR-A recognition.

The ubiquitin proteasome system (UPS) is essential in regulating myriad aspects of protein functions. It is therefore a fundamentally important regulatory mechanism that impacts most if not all aspects of cellular processes. Indeed, malfunction of UPS components is implicated in human diseases such as neurodegenerative and immunological disorders and many cancers. The success of proteasome inhibitors in cancer therapy suggests that modulating enzymes in the ubiquitination cascade would be clinically important for therapeutic benefits. In this review, we summarize advances in developing inhibitors of a variety of UPS components. In particular, we highlight recent work done on the protein engineering of ubiquitin as modulators of the UPS, a novel approach that may shed light on innovative drug discovery in the future.

Screening of a phage library displaying single chain fragments of the variable regions of human immunoglobulins (scFv) for binding to the ovarian chicken very low-density lipoprotein/vitellogenin receptor (OVR) led to the isolation of several antibody fragments with high affinity. As for the natural ligands of OVR, receptor binding of all antibody fragments is strictly Ca2+-dependent and is prevented by receptor-associated protein (RAP). Moreover, attachment of human rhinovirus serotype 2 (HRV2) to this receptor is inhibited by all scFvs. In contrast to conventional immunization, the in vitro selection method thus exclusively led to antibodies that attach to or close to the ligand binding site and thereby block the receptor-ligand interaction.

For more than 30years, the production of research antibodies has been dominated by hybridoma technologies, while modern recombinant technologies have lagged behind. Here I discuss why this situation must change if we are to generate reliable, comprehensive reagent sets on a genome-wide scale, and I describe how a cultural shift in the research community could revolutionize and modernize the affinity reagent field. In turn, such a revolution would pay huge dividends by closing the gap between basic research and therapeutic development, thus enabling the development of myriad new therapies for unmet medical needs.

Signaling via G-protein coupled receptors is initiated by receptor-catalyzed nucleotide exchange on Galpha subunits normally bound to GDP and Gbetagamma. Activated Galpha . GTP then regulates effectors such as adenylyl cyclase. Except for Gbetagamma, no known regulators bind the adenylyl cyclase-stimulatory subunit Galphas in its GDP-bound state. We recently described a peptide, KB-752, that binds and enhances the nucleotide exchange rate of the adenylyl cyclase-inhibitory subunit Galpha(i). Herein, we report that KB-752 binds Galpha(s) . GDP yet slows its rate of nucleotide exchange. KB-752 inhibits GTPgammaS-stimulated adenylyl cyclase activity in cell membranes, reflecting its opposing effects on nucleotide exchange by Galpha(i) and Galpha(s).

The CC chemokine, monocyte chemoattractant protein-1 (MCP-1), plays a crucial role in the initiation of atherosclerosis and has direct effects that promote angiogenesis. To develop a specific inhibitor for MCP-1-induced angiogenesis, we performed in vitro selection employing phage display random peptide libraries. Most of the selected peptides were found to be homologous to the second extracellular loops of CCR2 and CCR3. We synthesized the peptide encoding the homologous sequences of the receptors and tested its effect on the MCP-1 induced angiogenesis. Surface plasmon resonance measurements demonstrated specific binding of the peptide to MCP-1 but not to the other homologous protein, MCP-3. Flow cytometry revealed that the peptide inhibited the MCP-1 binding to THP-1 monocytes. Moreover, CAM and rat aortic ring assays showed that the peptide inhibited MCP-1 induced angiogenesis. Our observations indicate that the MCP-1-binding peptide exerts its anti-angiogenic effect by interfering with the interaction between MCP-1 and its receptor.

SurA is a periplasmic chaperone protein that facilitates maturation of integral outer membrane proteins (OMPs). Short peptides that bind SurA have previously been characterized. In this work, an enzyme-linked immunoabsorbent assay-based competition assay is utilized to demonstrate that binding of such peptides, presented by peptide-tagged phage, mimics binding of biological substrates. Two representative unfolded OMPs, OmpF and OmpG, bind SurA and a core structural fragment thereof in competition with peptide-tagged phage, and with the same order-of-magnitude affinity as the peptides. Additionally, unfolded OmpF and OmpG bind SurA more tightly than an unfolded water-soluble protein, while folded proteins have no measurable affinity, demonstrating a specificity of SurA for OMP polypeptides.

We report that, contrary to common belief, polypeptides fused to the carboxy-terminus of the M13 gene-3 minor coat protein are functionally displayed on the phage surface. In a phagemid display system, carboxy-terminal fusion through optimized linker sequences resulted in display levels comparable to those achieved with conventional amino-terminal fusions. These findings are of considerable importance to phage display technology because they enable investigations not suited to amino-terminal display, including the study of protein–protein interactions requiring free carboxy-termini, functional cDNA cloning efforts, and the display of intracellular proteins.

RNase S consists of two proteolytic fragments of RNase A, residues 1-20 (S20) and residues 21-124 (S pro). A 15-mer peptide (S15p) with high affinity for S pro was selected from a phage display library. Peptide residues that are buried in the structure of the wild type complex are conserved in S15p though there are several changes at other positions. Isothermal titration calorimetry studies show that the affinity of S15p is comparable to that of the wild type peptide at 25 degrees C. However, the magnitudes of DeltaH(o) and DeltaC(p) are lower for S15p, suggesting that the thermal stability of the complex is enhanced. In agreement with this prediction, at pH 6, the T(m) of the S15p complex was found to be 10 degrees C higher than that of the wild type complex. This suggests that for proteins where fragment complementation systems exist, phage display can be used to find mutations that increase protein thermal stability.

We have explored the possibilities of using human cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) as a single immunoglobulin fold-based scaffold for the generation of novel binding ligands. To obtain a suitable protein library selection system, the extracellular domain of CTLA-4 was first displayed on the surface of a filamentous phage as a fusion product of the phage coat protein p3. CTLA-4 was shown to be functionally intact by binding to its natural ligands B7-1 (CD80) and B7-2 (CD86) both in vitro and in situ. Secondly, the complementarity determining region 3 (CDR3) loop of the CTLA-4 extracellular domain was evaluated as a permissive site. We replaced the nine amino acid CDR3-like loop of CTLA-4 with the sequence XXX-RGD-XXX (where X represents any amino acid). Using phage display we selected several CTLA-4-based variants capable of binding to human αvβ3 integrin, one of which showed binding to integrins in situ. To explore the construction of bispecific molecules we also evaluated one other potential permissive site diametrically opposite the natural CDR-like loops, which was found to be tolerant of peptide insertion. Our data suggest that CTLA-4 is a suitable human scaffold for engineering single-domain molecules with one or possibly more binding specificities.

The recombinant phage antibody system pCANTAB 5E has been used to display functionally active leech-derived tryptase inhibitor (LDTI) on the tip of the filamentous M13 phage. A limited combinatorial library of 5.2×104 mutants was created with a synthetic LDTI gene, using a degenerated oligonucleotide and the pCANTAB 5E phagemid. The mutations were restricted to the P1–P4′ positions of the reactive site. Fusion phages and appropriate host strains containing the phagemids were selected after binding to thrombin and DNA sequencing. The variants LDTI-2T (K8R, I9V, S10, K11W, P12A), LDTI-5T (K8R, I9V, S10, K11S, P12L) and LDTI-10T (K8R, I9L, S10, K11D, P12I) were produced with a Saccharomyces cerevisiae expression system. The new inhibitors, LDTI-2T and -5T, prolong the blood clotting time, inhibit thrombin (Ki 302 nM and 28 nM) and trypsin (Ki 6.4 nM and 2.1 nM) but not factor Xa, plasma kallikrein or neutrophil elastase. The variant LDTI-10T binds to thrombin but does not inhibit it. The relevant reactive site sequences of the thrombin inhibiting variants showed a strong preference for arginine in position P1 (K8R) and for valine in P1′ (I9V). The data indicate further that LDTI-5T might be a model candidate for generation of active-site directed thrombin inhibitors and that LDTI in general may be useful to generate specific inhibitors suitable for a better understanding of enzyme-inhibitor interactions.

Phage antibodies have been widely prospected as an alternative to the use of monoclonal antibodies prepared by traditional means. Many monoclonal antibodies prepared against peptides are able to recognise the native proteins from which they were derived. Here we show that the same is also true for phage antibodies. We have selected a number of single-chain variable fragments (scFv) from a large phage scFv library against a peptide from the switch region II of p21Ras. This peptide is known to reside in a mobile area of the native protein and is the epitope of a well characterised monoclonal antibody. Selected scFvs were able to recognise native p21Ras in both ELISA and Western blots, indicating that peptides are also likely to be very useful in selecting from phage antibody libraries.

A random phage heptapeptide library was screened with rabbit antibodies against wheat flour proteins comprising gliadins and a small amount of low molecular weight glutenins (gli/glu). Gli/glu antibodies isolated from the sera selected different consensus sequences (CS). All CS contained tri- to pentapeptide stretches homologous to gli/glu sequences (proposed epitopes). In alpha- and gamma-type gliadins, these sequences are clustered in the N-terminal region recently suspected to be toxic for humans with celiac disease. Peptides with CS were synthesized and checked for reactivity. Only immune and no control rabbit sera reacted with synthetic peptides. One of eight human sera containing gliadin antibodies was reactive as well (4/8 peptides) but control sera were negative. Thus the phage display technique is useful for epitope screening of polyclonal antibodies even in the case of a group of homologous but diverse antigens.

Adequate means for genotype assignment to phenotype is essential in evolutionary molecular engineering. In this study, construction of 'in vitro virus' was carried out in which a genotype molecule (mRNA) covalently binds to the phenotype molecule (protein) through puromycin on the ribosome in a cell-free translation system. Bonding efficiency was approximately 10%, thus indicating a population of the in vitro virus to have approximately 10(12) protein variants, this number being 10(4) that in the phage display. The in vitro virus is useful for examining protein evolution in a test tube and the results may possibly serve as basis for a general method for selecting proteins possessing the most desirable functions.

Human antibodies specific for digoxigenin, estradiol, testosterone and progesterone have been isolated from a small combinatorial IgM repertoire (4×107) of single chain antibodies (scFv). The affinities of both the anti-estradiol and anti-progesterone scFv were approximately 108 M−1. Naive IgM genes appeared to be highly represented, since only the heavy chain variable domain of the anti estradiol antibody contained differences to corresponding germline sequences. The light chain variable domain of the progesterone receptor was also identical to a germline sequence, showing that it is possible for completely naive antibodies to bind steroids with affinities comparable to those obtained after a secondary immune response.

Vitronectin is a multifunctional serum protein which provides a unique regulatory link between cell adhesion, humoral defense mechanism and the hemostatic system, and the heparin-binding properties of vitronectin are thought to have participated in various functional aspects. In addition to the carboxy-terminal glycosaminoglycan-binding motif, we report on two novel heparin-binding domains which were identified using phage display technique. One heparin-binding domain is located between amino acids Asp82 and Cys137 at the end of the connector region, while the other is in the second hemopexin-type repeat, between amino acids Lys175 and Asp219 of the vitronectin molecule. Our findings may shed new light to the activities of vitronectin and its binding to cells, which could not be explained solely on the basis of the known heparin-binding domain.

To isolate specific single-chain variable (scFv) fragments against dihydroflavonol 4-reductase (DFR) from Petunia hybrida the phage display technology was used. DFR was overproduced in Escherichia coli, purified and used for immunization. From DFR-immunized mice, a phage display library was made starting from spleen mRNA using an optimized set of primers for VH and VL amplification. Several rounds of panning against recombinant DFR yielded five different scFv fragments, confirmed by subsequent DNA sequencing. They all specifically bound to recombinant DFR in ELISA and DFR in flower extracts on Western blot. These results show that phage display is a promising technology in plant molecular biology to obtain specific recombinant antibodies not only for ELISA and Western blot but also for in vivo applications in the long run. © 1997 Federation of European Biochemical Societies.

In order to establish a subtractive procedure that makes it possible to enrich selectively phage displayed antibodies directed against proteins constituting a difference between two populations of cells, a competitive selection strategy utilising two solid phases was developed and tested. Antibodies recognising a defined difference between two otherwise identical protein mixtures were isolated and their specificity confirmed. To test further the efficacy of selection inhibition during the competitive selections, selections towards a total cell extract were performed with and without competition from the same extract. An analysis of the resulting phage antibodies confirmed the subtractive nature of the system described.

Heterotrimeric (alpha beta gamma) guanine nucleotide binding proteins (G-proteins) dissociate into their constituent subunits in the course of signal transduction. Exposure of the G-protein G(o) to the alpha(o)-specific monoclonal antibody 3E7 results in recovery of alpha(o) alone. We identified the 3E7 epitope as ERSKAIEKNL (positions 14-23) using synthetic peptides and phage display. G(o) isolated with alpha(o)-specific monoclonal antibodies MONO and 3C2 dissociates and releases its beta gamma subunits when exposed to 3E7. Exposure to 3E7, but not MONO or 3C2, results in the displacement of beta gamma from trimers, in the absence of added activators of G-proteins (GTPgammaS, Mg2+AlF4-). We propose that stable binding of 3E7 to alpha(o) requires displacement of beta-gamma and occurs in the absence of guanine nucleotide exchange.